In this experiment HEK293T derived EVs overexpressing CD63 dTomato were simultaneous fluorescently labeled with CD63 APC and CD81 BV421. Labeling and fluorescence NTA (F-NTA) measurements were done immediately after the sample was thawed. Additional aliquots were stored at 25°C for 3, 6, and 10 days after thawing, followed by labeling and F-NTA analysis.
Within the 10 day experimental timeframe, a substantial reduction of EV concentration was observed in F-NTA via CD63 detection (Fig. 2). The labeling efficiency of CD63 APC compared with intrinsic CD63 dTomato labeling was, for any given day, usually near 100%. The stepwise reduction in CD63 dTomato and CD63 APC concentrations are nearly identical over the observed time intervals. These results indicate reliability in the experiments as well as compatibility between CD63 dTomato and further labeling with CD63 APC antibody. However, CD81 BV421 did not show any reduction over time.
A significant change in size, which is sometimes measured after thawing, was not observed, and is likely dependent on the membrane properties such as rigidity/fluidity or protein content of the EVs. However, it can be assumed that a constant degradation of vesicles, over our storage time intervals, was responsible for the reduction in EV counts measured via scatter and F-NTA.
Conclusion
The results show that storage of EVs at 25°C results in a constant and ongoing decrease in particle count in scatter NTA as well as a reduction in CD63 expressing vesicles, indicating that the observed decrease in the overall particle number is caused by a degradation of the vesicles.Interestingly, the concentration of CD81 expressing vesicles remains stable over time, giving a hint that this subpopulation might be more stable under the given storage conditions.Concentration measurements via scatter and F-NTA can provide a quick and reliable quality assessment. Size, concentration and fluorescence can be quickly measured on one EV sample in a single run on the ZetaView®.
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